The instant invention relates to an altered flower shape in plants belonging to the genera Osteospermum and Dimorphoteca, which is induced by a mutant allele, as well as to the method of breeding Osteospermum and Dimorphoteca plants having this altered flower shape.
The genus Osteospermum was introduced as a commercial bedding plant in the beginning of the nineties of the last century. Since then this genus has been very successful in the horticultural market. For 2008, worldwide sales were estimated at almost 100 million plants.
The genus Osteospermum is a South African native and belongs to the plant family of the Asteraceae. It comprises almost 70 different species representing a broad range of either evergreen shrubs or herbaceous plants with growing habits varying from erect to prostrate. The existing Osteospermum cultivars are thought to be interspecific hybrids of the following main species: O. ecklonis, O. barbariae, O. caulescens, O. fruticosum, O. jucundum, and O. chrysanthemifolia. The first breeding with Osteospermum was started between 1970 and 1985 by British hobby breeders and later continued mainly by Danish and Japanese breeders (Allavena, A., et al., Genetic engineering of Osteospermum ssp.: a case story, Acta Hort., 508, 129-133 (2000)). According to Faccioli, et al., professional breeders used the British plant material as well as accessions from South Africa to breed new hybrids (Faccioli, P., et al., Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis, Plant Breed., 119, 351-355 (2000)). During further breeding, which was mainly done by professional Danish and German breeding companies, crossings between the existing plants were made to improve the quality. This approach has resulted in a narrow gene pool of the plant material which is commercially available today. The genera Osteospermum and Dimorphoteca are very closely related and, in some cases, even the distinction of both genera or the classification of certain varieties into these two genera is unclear. In the past the genus Osteospermum belonged to the genus Dimorphoteca, but today Dimorphoteca only comprises the annual species, whereas all semi-perennial species fall into the genus Osteospermum. Crossbreeding between both genera is possible and several commercial varieties result from intergeneric hybridization between an Osteospermum and a Dimorphoteca parent. The different Osteospermum and Dimorphoteca cultivars, breeding lines and wild species represent a broad range of different ploidy levels varying from 2× up to almost 8×, which also shows that during the development of today's cultivars hybridization between species took place. For commercial production, Osteospermum and Dimorphoteca plants are mostly propagated asexually by cuttings. However, sexual propagation through seeds is also possible and several seed propagated varieties are on the market.
Commercially available Osteospermum plants flower from early spring to autumn. The typical flower is a capitulum (flower head) with tubular central disc florets surrounded by a ring of ray florets, which gives the flowers the typical daisy shape (Faccioli, P., et al., Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis, Plant Breed., 119: 351-355 (2000)). The color and shape of ray florets as well as the color of the disc florets vary. The color of the upper surface of the ray florets, which in colloquial language are called petals, is determined by two independent metabolic pathways producing carotenoids, visible as yellow-orange-brown colors, and anthocyanins, resulting in white to pink and purple flower colors (Seitz, C., Klonierung and Charakterisierung von Flavonoidgenen aus Osteospermum, Dissertation an der Technischen Universität München (2004)). Intensive breeding work during the past several years has resulted in a broad range of white, pink, purple, yellow, and orange petal colors and new mixes of the carotenoid and anthocyanin color groups, as well as in color patterns like eye types or stripes. Similar to the color range of the upper surface, the color of the lower surface of the ray florets also varies from light to dark colors in the bluish-pink or yellow-brown color range. The color pattern usually is striped with the colored stripes running parallel to the petal edges. Typically, the color of the disc florets is darker than the color of the ray florets and it may vary from grey to blue, violet or purple or from dark yellow to dark brown. The usual shape of the ray floret is obovate, but in some genotypes the petal edges are rolled upwards resulting in so-called spoon or spider types.
An Osteospermum and Dimorphoteca breeding program was established in 2002 to produce altered flowering Osteospermum and Dimorphoteca plants. Osteospermum plants with unusual inflorescences are desirable, as altered flowers, which display mainly enlarged disc florets, are believed to stay open even in complete darkness, whereas normal flowers close under low light conditions (less than 2000 Lux).
Furthermore, for the altered flowering plants, the keepability of the flowers is longer both in the field and in the greenhouse compared to the flowers on a normal-flowering plant. This extended flower keepability is believed to result from a reduced seed set due to the limited pollen availability on the altered flowering plants. This limited pollen availability is a direct consequence of the enlarged disc florets which prevent the pollinating insects from reaching the pollen.
Finally, although most commercially available Osteospermum and Dimorphoteca varieties or assortments are vegetatively propagated by cuttings, several varieties or assortments of the genera Osteospermum and Dimorphoteca such as “Asti” and “Passion Mix” are propagated by seeds. For the production of F1 hybrids of the seed-propagated varieties, the flowers of the female crossing parent usually have to be emasculated to prevent self-pollination and then are pollinated with pollen of a selected male parent to produce hybrid seeds. To avoid the labor intensive and costly emasculation and hand pollination procedures, a system that inhibits self pollination on the bisexual Osteospermum and Dimorphoteca plants would be highly desirable. In some plant species biological systems like male sterility or self incompatibility can be used in this respect, but these systems are not described for Osteospermum or Dimorphoteca. However, in case of Osteospermum and Dimorphoteca plants, which exhibit the altered flower type with enlarged disc florets, anthers are covered by the enlarged disc florets and hence the pollen is not freely available for pollinating insects. Therefore, hybrid seeds from these plants can be obtained without emasculation by insect pollination, which reduces the costs for F1 hybrid seed production significantly.
For the above reasons, it is desirable to develop altered flowering Osteospermum and Dimorphoteca plants which show enlarged disc florets.
Several approaches such as mutation treatment and interspecific and intergeneric crosses were attempted to achieve an altered flowering trait in the genera Osteospermum and Dimorphoteca. 
Several experiments on induction of mutations by Gamma-irradiation of Osteospermum and Dimorphoteca plant material were performed. Examples of references that illustrate alteration of flower type via mutation are altered flower type in ornamental sweet potato (Bhate, R. H., Chemically Induced Floral Morphological Mutations in Two Cultivars of Ipomoea purpurea (L.) Roth, Scientia Horticulturae 88: 133-145 (2001)); in Chrysanthemum (Rana, R. S., Radiation-Induced Variation in Ray-Floret Characteristics of Annual Chrysanthemum, Euphytica. 8: 270-322 (1965)); in roses (Teruo, N., Ikegami, Y., Matsuda, Y., and Toyoda, H., Induction of Morphologically Changed Petals from Mutagen-Treated Apical Buds of Rose and Plant Regeneration from Varied Petal-Derived Calli, Plant Biotechnology, 8: 233-236 (2001)); and in plants in general (Krasaechai, A. L. D., et al., Low-Energy Ion Beam Modification of Horticultural Plants for Induction of Mutation, Surface and Coatings Technology, 203: 2525-2530 (2009)). In this regard it is important to mention that the ploidy level of almost all Osteospermum cultivars is tetraploid, whereas the ploidy level of Dimorphoteca cultivars varies from 2× to 6×. This means that in the case of a recessive mutation at least two generations would be necessary for the phenotype of any recessive mutation to become visible. In the case of a dominant mutation the phenotype would become visible in the M0-generation.
A first set of experiments was performed by the applicant on mature seeds which had been harvested from different Dimorphoteca cultivars. Batches of 30 seeds each were treated with doses of Gamma-irradiation varying from 15 to 40 Gy for periods varying from 5 to 30 minutes. Immediately after this treatment the seeds were soaked in a solution of 10% Polyethylene glycol (PEG) for 4 hours, the solution was washed off and the seeds were sown in standard seedling substrate. Germination started after about one week. Three weeks after sowing, when the first pair of leaves had developed, the seedlings were transplanted. Three weeks after transplanting, the seedlings were planted into 11 cm diameter pots and grown according to standard protocols. First flowering started about 10 weeks after potting. The plant populations were continuously evaluated for effects or mutants caused by the Gamma-irradiation. Depending on the dosage and the period of irradiation fewer seeds germinated and more malformed seedlings appeared, which did not develop further. Alterations of the growing habit as well as altered foliage types were difficult to evaluate, because the seeds originated from crossbreeding and therefore segregation of these characters in the offspring was expected. However, altered flower colors appeared, which resulted from mutation and not from segregation of the parental flower colors. These new colors showed that overall mutations of flower characteristics had successfully been induced by Gamma-irradiation. However, no altered flower shapes were detected in these plant populations.
A second set of experiments was performed by the applicant on rooted cuttings from different Dimorphoteca cultivars. Cuttings were rooted in standard paper pots within a period of 4 weeks. After successful rooting the cuttings were pinched above the 5th leaf pair and immediately Gamma irradiated. The dosages and irradiation periods corresponded to the previous experiments on seeds. After irradiation the cuttings were planted into 11 cm diameter pots containing a standard growing substrate and cultivated under standard growing conditions. The young plants were pinched back twice over a period of 6 weeks in order to allow mutated cells to develop into shoots. Flowering started about 13 weeks after planting. The plants were continuously evaluated for mutants. Several altered growing habits, foliage shapes, and flower colors were detected. However, altered flower shapes did not appear on any of the irradiated plants.
Interspecific and Intergeneric Crosses
Representatives from different species of the genera Osteospermum and Dimorphoteca were collected and crossing experiments with commercial Osteospermum cultivars were performed. In all combinations one parent was a commercial variety.
Occasionally, in Osteospermum seedling progeny individual plants were detected which exhibited an additional whorl of ray florets. These florets, which were located at the base of the main ray florets, were significantly narrower than the main ray florets and orientated vertically to the first whorl. These flowers still produced female organs at the base of the ray florets and were female fertile which was proven by their seed set. This additional whorl of ray florets was not stable and showed significant genotype-environment interaction. The respective plants were self-pollinated as well as crossbred to stabilize this phenotype. However, the trait was not detected in any of the progeny and therefore it is obviously not genetically stable. In summary, interspecific or intergeneric seed set was achieved for only two combinations, which was shown by an intermediate phenotype of the offspring. Among this offspring, as well as in further generations produced from these plants, no stable altered flowering plants were detected.
Hence, there is still a need for stable altered flowering plants having enlarged disc florets. The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.